Alternate CD4/CD8 lineage commitment is determined by differential expression of ThPOK, which is in turn controlled at the level of transcription. We postulate that differential ThPOK expression is regulated instructively by TCR signaling. Our long- term goal is to define the pathways linking TCR signaling with differential ThPOK expression. Preliminary results show that ThPOK expression is controlled by 2 alternate promoters, which are preferentially activated at different stages of thymic development. The present proposal pursues three complementary aims to elucidate the connection between TCR signaling and ThPOK transcription. Aim 1: Non-overlapping reporter cassettes centered on the alternate distal and proximal promoters show preferential expression in CD4 thymocytes, indicating the existence of 2 different sets of regulatory elements that can mediate CD4 lineage-specific expression. In this aim, we will 1. map important transcriptional control elements using in vitro and in vivo reporter assays, 2. define changes in chromatin accessibility at the ThPOK locus during development using the DNase I hypersensitivity (DHS) approach, and 3. identify candidate regulatory factors for the distal cassette and test their functional significance. Aim 2: We hypothesize that distal and proximal promoters may control transient TCR-dependent induction and long-term CD4 lineage-specific expression of ThPOK, respectively. In this aim we will 1. Test dependence of distal and proximal reporter cassettes on TCR signaling or CD4 lineage-specific control mechanisms using mice in which only one or the other mechanism is active, 2. Determine the functional requirement for each promoter in CD4 commitment, using knockin mice in which one or the other promoter is specifically inactivated, and 3. Compare ThPOK expression at the single-cell level between thymocytes of different specificities and affinities, using single-cell PCR and reporter knockin mice, to test key aspects of the competing signal strength and kinetic signaling models of lineage commitment. Aim 3: ThPOK and Gata-3 are both required for CD4 commitment, but their functional relationship remains unclear. We will test whether ThPOK overexpression can correct the developmental deficiency in Gata-3-/- mice (and vice versa), whether Gata-3 is required prior to ThPOK upregulation, and whether Gata-3 is directly involved in regulation of ThPOK transcription. Project Narrative: The mechanisms that control lineage commitment of CD4 and CD8 T cells are critical to development of a normal immune system and therefore essential to health. Furthermore, we have demonstrated that ThPOK is a potent inducer of T cell lymphomagenesis in mice, and is overexpressed in a substantial fraction of human T cell lymphomas. Hence understanding the control of ThPOK expression is also directly relevant to the mechanism of lymphomagenesis.